DESCRIPTION (Principal Investigator's Abstract): The long range goal is to examine the genetic basis for selenocysteine (SeCys) incorporation into animal and bacterial proteins. The specific aims are to purify and sequence the low molecular weight selenium (Se)- containing protein, called the G protein; clone and sequence the G protein gene; clone and sequence phylogenetically diverse genes for glutathione peroxidase (GPX) and identify the G protein and GPX DNA sequences; use mutational analysis to define regions of the formate dehydrogenase gene essential for SeCys incorporation; and to use gene-fusion experiments to study SeCys incorporation into peptides. It is proposed to use monoclonal antibodies to purify the G protein, to determine the G protein content in various tissues of rats as affected by Se status, and to clone the G protein gene. The sequences of amino acids around SeCys of the selenopeptide form the G protein will be determined to develop a synthetic probe and to compare to that for GPX. A phylogenetic comparison of GPX DNA sequences from nonmammalian to mammalian species will be made by cloning genes from chickens, trout and house fly. In the bacterial work, the expression of fusion genes coding for chimeric proteins consisting of the SeCys coding regions of either the E, coli formate dehydrogenase gene or bovine GPX to the B galactosidase gene will be examined in both the E, coli and mammalian transient expression systems. Once it has been confirmed that bacterial systems can direct synthesis of a selenopolypeptide with B galactosidase activity, work will be done to isolate mutants which have lost the ability to incorporate SeCys through cis action mutations. Results should be obtained to determine the mechanism whereby UGA codon directs SeCys incorporation rather than serving as a stop codon. Using the information obtained from the bacterial work, the expression of the same constructs will be examined by transient expression in mammalian cells. A better understanding of the metabolis of Se should provide information on the relationship of this element to metabolic disorders such as cancer or heart disease (Keshan disease).